Translation of the Alzheimer Amyloid Precursor Protein mRNA Is Up ...

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APP evidently can cause AD because all individuals with Down syndrome have an extra copy of the APP gene on chromosome. 21 and invariably develop AD ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 274, No. 10, Issue of March 5, pp. 6421–6431, 1999 Printed in U.S.A.

Translation of the Alzheimer Amyloid Precursor Protein mRNA Is Up-regulated by Interleukin-1 through 5*-Untranslated Region Sequences* (Received for publication, September 23, 1998, and in revised form, December 1, 1998)

Jack T. Rogers‡§, Lorene M. Leiter‡¶, Jay McPhee‡i, Catherine M. Cahill**, Shan-Shan Zhan‡‡§§, Huntington Potter‡‡, and Lars N. G. Nilsson‡‡¶¶ From the ‡Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, the **Diabetes Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, and the ‡‡Department of Biochemistry and Molecular Biology and Suncoast Gerontology Center, University of South Florida, Tampa, Florida 33612

The amyloid precursor protein (APP) has been associated with Alzheimer’s disease (AD) because APP is processed into the b-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1a and IL-1b increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5*-untranslated region (5*UTR) conferred translational regulation by IL-1a and IL-1b to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5*UTR)/CAT mRNAs were unchanged, whereas both baseline and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from 155 to 1144 nucleotides from the 5*-cap site and is homologous to related translational control elements in the 5*-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.

The amyloid precursor protein (APP)1 is a 110 –130-kDa type * This work was supported in part by National Institutes of Health Grant AR29I32717, American Federation for Aging Research Grant 96022, Alzheimer’s Association Grant PRG-94-146, a pilot grant from the Alzheimer’s Disease Research Center, Boston (to J. T. R.), and a National Institutes of Health Grant AG09665 (to H. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: 604 LMRC, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0370; Fax: 617-7393324; E-mail: [email protected] ¶ Recipient of Postdoctoral Fellowship 6872302 from the NCI, National Institutes of Health. i Present address: Dept. of Genetics, Harvard Medical School, Boston, MA 02115. §§ Present address: Schepens Eye Institute, 20 Staniford St., Boston, MA 02114. ¶¶ Recipient of postdoctoral fellowships from the Swedish Foundation for International Cooperation in Research and Higher Education and Riksbankens Jubileimsfond (following a donation from Erik Ro¨nnberg). 1 The abbreviations used are: APP, amyloid precursor protein; Ab, b-amyloid; AD, Alzheimer’s disease; ACT, a1-antichymotrypsin; IL-1, interleukin-1; apoE, apolipoprotein E; UTR, untranslated region; L, light; H, heavy; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; GFAP, glial fibrillary acidic protein; bp, base pair(s); kb, kilobase(s); GAPDH, glyceraldehyde-3-phosphate dehydroThis paper is available on line at http://www.jbc.org

I membrane-spanning glycoprotein expressed ubiquitously in mammalian tissues (1, 2). A portion of APP is processed constitutively into 40 – 42-amino acid b-amyloid (Ab) peptides, which then polymerize and deposit as the amyloid filaments as one of the pathological hallmarks of Alzheimer’s disease (AD) and Down syndrome (3–7). The regulation of APP gene expression as a pathogenic factor for AD has received considerable attention. Several putative physiological activators of APP gene transcription have been defined (8 –10). Overexpression of APP evidently can cause AD because all individuals with Down syndrome have an extra copy of the APP gene on chromosome 21 and invariably develop AD pathology by the age of 40 –50 years (11, 12). In addition to altered Ab cleavage, secretion, and deposition (5), accumulating evidence has revealed that local inflammation at the site of developing extracellular plaques in the brain is important to AD pathogenesis (13–15). For example, a1antichymotrypsin (ACT) is present in amyloid plaques, and its production by adjacent astrocytes suggests the occurrence of an inherent inflammation in the AD brain, similar to the hepatic acute phase response (16, 17). Other significant markers, such as interleukin-1 (IL-1)-positive microglia and complement protein, confirm the presence of local inflammatory events during AD progression (18, 19). Epidemiological studies identifying traumatic head injury as a risk factor for AD strengthen the hypothesis that inflammatory mechanisms contribute to the disease pathogenesis (20). Hippocampal lesion has been shown to increase APP immunoreactivity in neighboring astrocytes (21). In vitro studies, and recently an in vivo study, have shown that certain proteins, e.g. ACT and apolipoprotein E (apoE), which are expressed during traumatic injury and inflammation of the brain parenchyma, might regulate the polymerization of Ab peptides into amyloid filaments (22–27). IL-1 is the first proinflammatory cytokine secreted after the activation of macrophage/microglial cells (28). IL-1 is expressed abundantly in microglia around developing amyloid plaques in brain cells, particularly in those brain regions that are prone to develop the mature amyloid plaques enriched in b-sheet protein structure (17, 18, 29). IL-1 action is mediated by two separate cytokines, IL-1a and IL-1b, which share low sequence homology (30%) and are encoded by two separate genes derived from a common ancestor gene (30). IL-1a and IL-1b target the same signaling receptor and exert overlapping proinflammatory effects, although the processing and site of action of these cytokines differ (28).

genase; APP(s), secreted APP; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid.

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Similarities in APP mRNA and Ferritin mRNA Translational Control

Microglial IL-1, which is known as a stimulator of astroglial proliferation (31), is increased in the rat brain after injury (32). A protein kinase C-dependent pathway (9) has linked IL-1 to a 3-fold increase in APP gene expression in human endothelial cells at the level of enhanced transcription. IL-1 greatly increased the transcriptional regulation of the amyloid plaqueassociated protein ACT in human primary astrocytes (17) and a human astrocytoma cell line (33). However, a smaller induction of APP gene transcription by IL-1 in rat neuronal cells was not matched by an increase in the steady-state levels of APP mRNA in glial cells, including astrocytes (2, 34). In this report we have identified and characterized a novel mechanism of IL-1-dependent regulation of APP gene expression at the level of increased APP mRNA translation in astrocytes. Here, increased synthesis of APP by IL-1 was found to be mediated through a translational regulatory sequence in the 59-untranslated region (59-UTR) of APP mRNAs. We showed previously that IL-1 specifically enhances translation of the mRNAs encoding the light (L) and heavy (H) subunits of ferritin, the central iron storage protein present in all cells (35). The APP mRNA 59-UTR sequence can fold into a single RNA stemloop and is related to hepatic RNA enhancers, the acute box sequences, in the 59-UTR of L- and H-ferritin mRNAs (36, 37). EXPERIMENTAL PROCEDURES

Cell Culture and Immunocytochemistry Primary human astrocytes were prepared by trypsinization of human fetal brain tissue as described previously (17), treatment with 5 mM H-Leu-O-methyl ester to eliminate microglia (38), seeding onto coated plates, and growing to 70% confluence in DMEM (low glucose) supplemented with 10% fetal bovine serum. For immunohistochemistry the cells were grown on 10 mg/ml poly-L-lysine-coated microscope slides, washed briefly in 1 3 PBS, and then fixed in 4% paraformaldehyde and 0.1% Triton X-100 in 1 3 PBS, pH 7.4, for 30 min on ice. The cells were then incubated in 10% normal goat serum (Life Technologies, Inc.) and 0.4% Triton X-100 in 1 3 PBS for 30 min at 37 °C to block nonspecific binding. The primary antibody (monoclonal anti-glial fibrillary acidic protein (GFAP), clone G-A-5, Sigma, dilution 1:400), was applied in 1% normal goat serum and 0.4% Triton X-100 in 1 3 PBS for 1 h at 37 °C. The astrocytes were washed for 5 min in 1 3 PBS and incubated with the secondary antibody (affinity-purified polyclonal Cy3-labeled goat anti-mouse IgG (H1L), Jackson ImmunoResearch, dilution 1:400) in 10% normal goat serum and 0.4% Triton X-100 in 1 3 PBS for 30 min at room temperature. The cells were again washed in 1 3 PBS for 5 min, counterstained with 4 ng/ml 4,6-diamidino-2-phenylindole, mounted in 50% glycerol, and examined with a light microscope (Axioskop, Zeiss). The U373MG astrocytoma cell line was obtained from Dr. H. Fine (DFCI, Boston, MA) and cultured on uncoated dishes to 60 – 80% confluence in DMEM (high glucose) supplemented with 10% fetal bovine serum.

Plasmid Constructs The eukaryote expression vector pSV2CAT contains a unique StuI site 45 base pairs (bp) downstream from the SV40 early T-antigen promoter. A unique HindIII site is present 17 bp further downstream from the StuI site (62 bp from the CAT gene 59-cap site (36)). The pSV2(APP)CATconstruct was prepared by inserting 90 bp of the APP gene 59-UTR (between the SmaI and the NruI sites, respectively 155 and 1144 nucleotides from the 59-cap site) into the StuI and HindIII sites in the 59-UTR of the CAT gene in pSV2CAT (see Fig. 6). This pSV2(APP)CAT construct was prepared by two steps of subcloning because the APP gene 59-UTR is GC-rich and was refractory to accurate amplification by polymerase chain reaction. First, a 3-kb SmaI-HindIII fragment containing the APP gene was subcloned into compatible StuI and HindIII sites unique to the 59-UTR of the CAT gene in the pSV2CAT expression vector. The fragment between the NruI and HindIII sites in the APP gene was then removed from the construct. The restriction sites were then blunt ended and religated. The pBS2CAT construct contained a 254-bp HindIII-EcoRI fragment from the CAT gene coding for 218 bp of coding sequences from CAT gene ligated immediately downstream from 36-bp 59-UTR sequences from the SV40 early Tantigen promoter region. These sequences from the CAT gene were ligated into the unique polylinker site in the pBS vector (Stratagene).

The pGEM3zF6-hACT, pGEM3zF6-hAPP, and pGEM3zF6-hGAPDHconstructs, respectively, contained a 407-bp PstI-SacI fragment (amino acids 175–311 in the human ACT gene (39)), a 1,056-bp EcoRI-EcoRI fragment (bp 1795–2856 in the human APP gene (40)), and a 548-bp HindIII-XbaI fragment (amino acids 66 –248 in the human GAPDH gene (41)). These inserts were subcloned into the pGEM3zf1vector (Stratagene). Restriction-digested DNA from these constructs was used as a template to synthesize antisense cRNAs.

APP Synthesis Primary Astrocytes—GFAP-positive astrocytes (1 3 105 cells/well) and astrocytoma cells (70% confluent) were measured for intracellular APP synthesis and ferritin synthesis after stimulation with 0.5 ng/ml recombinant IL-1a (Genzyme), 0.5 ng/ml IL-1b (Genzyme), 10 mM ferrotransferrin (Boehringer Mannheim), 100 mM desferrioxamine (Ciba Geigy), or left untreated as controls. Cell numbers from individual wells were counted to ensure that 1 3 105 cells were present in each well at the beginning of each labeling experiment. Astrocytes were preincubated for 15 min in methionine-free medium (RPMI 1640; Life Technologies, Inc.) and pulse labeled for 30 min with 300 mCi/ml [35S]methionine. Each microtiter plate was washed twice in cold PBS at 4 °C before lysis of astrocytes in 25 ml of STEN buffer (0.2% Nonidet P-40, 2 mM EDTA, 50 mM Tris, pH 7.6) using a sterile glass rod. L-Phenylmethylsulfonyl fluoride (100 mg/ml) and leupeptin (2 mg/ml) were added to the STEN lysis buffer to prevent proteolysis. Half of the pooled lysates (i.e. 300-ml total volume from each row of 12 wells) were immunoprecipitated with a 1:500 dilution of a COOH-terminal directed APP antibody (C-8, 1:500 dilution, against amino acids 676 – 695 of APP-695; gift from D. Selkoe (42)). The other half of the lysates were immunoprecipitated with human ferritin antiserum (1:500 dilution, Boehringer Mannheim). Labeling of secreted APP (APP(s); Protease-Nexin-2) was measured after 2 h of pulse labeling astrocytes with 300 mCi/ml [35S]methionine, after which 2 ml of medium was precleared by centrifugation in Eppendorf tubes (10,000 rpm for 10 min), and the supernatant was immunoprecipitated (1:1,500 dilution, against amino acids 595– 611 of APP, R1736, gift from D. Selkoe). ApoE was immunoprecipitated from both the lysate and the medium using a 1:200 dilution of a polyclonal antiserum (Chemicon). Astrocytoma Cells—U373MG astrocytoma cells (80% confluent) were stimulated with IL-1a and IL-1b at concentrations between 0.05 and 5 ng/ml. After stimulation, equal numbers of cells were labeled for 30 min with 300 mCi/ml [35S]methionine in DMEM lacking methionine, washed twice with PBS, and the cell pellets were lysed in 200 ml of cold STEN buffer containing 100 mg/ml phenylmethylsulfonyl fluoride. There were 2 3 107 cells/10-cm2 plate at the beginning of each pulse labeling (see Fig. 4), and 2 3 106 cells were present in each well of six-well plates in the experiment described in Fig. 5. The protein concentrations of each lysate were measured to confirm that equal numbers of cells had been pulse labeled. Total protein synthesis was measured by the amount of [35S]methionine incorporation using 10% trichloroacetic acid and 2% casamino acids to precipitate labeled proteins present in each lysate (4 °C). Triplicate samples (10 ml) were assayed after hydrolysis of methionine charged tRNAs with 250 ml of 1 M NaOH and 1.5% H2O2 at 65 °C for 30 min. APP and ferritin were immunoprecipitated from U373MG astrocytoma lysates by adding 2 ml of anti-APP antibody (C-8 antibody) (42) as described for primary astrocytes. Immunoprecipitations—In all labeling experiments, antigen-antibody complex was collected with protein A-Sepharose beads (Pierce) and the immunoprecipitates applied to 10 –20% Tris-Tricine gels (Novex) and fractionated by electrophoresis in a buffer containing 0.1 M Tris-Tricine buffer and 0.1% SDS, pH 8.3. The gels were fixed with 25% methanol, 7% (v/v) acetic acid for 1 h, incubated with a fluorographic reagent (Amplify; Amersham Pharmacia Biotech) for 30 min, dried, and exposed to Kodak X-Omat film overnight at 280 °C.

RNA Purification and Northern Blot Hybridization Equal numbers of cells were pelleted by centrifugation at 1,000 rpm and the pellets lysed using 1 ml of modified guanidinium/phenol reagent according to the manufacturer’s instructions (Tri-reagent, MRC Research Inc., Cincinnati, OH). The RNA pellet was resuspended in TES buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 0.5% SDS, pH 7.6), and the A260/280 n was measured as an estimate of purity and RNA concentration. A cesium chloride procedure for the purification of RNA from DNA was followed for all experiments involving the use of transfected DNA (36). Total RNA samples from both transfected and untransfected cells were denatured in 50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA, pH 7.4, at 60 °C for 10 min. M

Similarities in APP mRNA and Ferritin mRNA Translational Control

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FIG. 1. GFAP-positive astrocytes. Human primary astrocytes displayed immunofluorescent GFAP staining using a monclonal antibody (clone G-A-5) and a Cy3-labeled goat anti-mouse secondary antibody (panel A) but very faint background staining following omission of primary antibody (panel B). Panels C and D, 4,6-diamidino-2-phenylindole staining of the same cells.

RNA samples were separated by electrophoresis on 1.5% agarose-formaldehyde gels, blotted onto Hybond-N filters, and immobilized by UV cross-linking (2 min) and heating of filters to 80 °C for 1 h. Filters were prehybridized overnight to reduce background hybridization signal and then hybridized overnight in 50% formamide, 50 mg/ml denatured salmon sperm DNA, 5 3 SSC, 0.1% SDS, and 5 3 Denhardt’s solution. After hybridization, filters were washed twice for 1 h in 2 3 SSC and 0.2% SDS at room temperature. Filters were washed further through four changes of 0.5 3 SSC and 0.1% SDS at 55 °C. In all experiments, equal loading of Northern gels was verified by ethidium bromide staining of 18 S and 28 S rRNAs and GAPDH hybridization as an internal standard (41). Specific RNAs were detected either by hybridization of Northern blot filters with randomly primed cDNA probes (35) or with copy RNA (cRNA) probes (37). Antisense cDNA hybridization probes were labeled by random primed labeling (specific activity 5 2 3 108 cpm/mg), and cRNA probes were transcribed in vitro with T7 RNA polymerase (Promega, Madison, WI) in the presence of [32P]CTP (3,000 Ci/mmol, NEN Life Science Products).

Transient Transfections and CAT Assays Transient Transfections—Astrocytoma cells (1 3 107 cells/10-cm2 plate) were transfected with 20 mg of DNA, each purified from the pSV2CAT or pSV2(APP)CAT constructs. Inclusion of RSV2-GAL (5 mg) in each transfection was used to standardize for differences in the transfection efficiency of the pSV2CAT and pSV2(APP)CAT constructs. Briefly, LipofectAMINE reagent (Boehringer Mannheim) was added to DMEM (without serum) and incubated for 45 min at room temperature. Plasmids (20 mg), in an equal volume of DMEM, were incubated for 45 min at room temperature with the LipofectAMINE reaction mixture. LipofectAMINE-DNA complex was then applied for 4 h to U373MG astrocytoma cells grown to 60% confluence. The cells were washed twice in DMEM (without serum), and subsequently cells were incubated for 24 – 48 h with DMEM supplemented with 10% fetal calf serum in the presence or absence of cytokine before harvesting and CAT assay. Transfected cells were stimulated with cytokine by the administration of 0.5 ng/ml IL-1a, 0.5 ng/ml IL-1b, or left as unstimulated controls. After defined times of cytokine stimulus (20 h in Fig. 7A), cells were harvested in PBS and assayed immediately for CAT activity or CAT mRNA levels (Northern blotting). CAT Assays—After harvesting, equal numbers of cells from 10-cm2 tissue culture plates were resuspended in 1 ml of 0.25 M Tris, pH 7.8, and subjected to three cycles of freezing and thawing (liquid nitrogen, 37 °C) to lyse the cells. Lysates were collected after centrifugation at 10,000 rpm for 5 min. The protein concentration was measured using the manufacturer’s conditions (Bio-Rad assay). Lysates (20 mg of protein) were added to 50 ml of a CAT reaction mix containing 1 M Tris, pH 7.8, 20 ml of acetyl-CoA (3.5 mg/ml), and 5 ml of 14C-labeled chloramphenicol (25 mCi/ml). After incubation for 1 h at 37 °C, reaction products were extracted with 1 ml of ethyl acetate, and the samples were resolved by thin layer chromatography as described previously (37). Marked areas on the TLC plate were excised and quantified by scintil-

lation counting (Econofluor, NEN Life Science Products) using a Wallac 1409 counter (Amersham Pharmacia Biotech). The average CAT activity from a number of separate transfection experiments (see Fig. 7B) includes CAT activity as estimated by use of an assay directly counting the amount of CAT reaction product partitioning into liquid scintillation fluid (37). Galactosidase Assay—Lysates were diluted at equal protein concentration in a CAT lysis buffer (Promega). Extracts were then incubated in the presence of ONPG color dye, and b-galactosidase enzyme activity was determined according to the manufacturer’s conditions. RESULTS

IL-1 Stimulates APP Gene Expression at the Translational Level in Primary Human Astrocytes—APP synthesis was measured in primary human fetal brain astrocytes (95–100% GFAPpositive cells in culture (Fig. 1)) after treatment with both IL-1a and IL-1b (0.5 ng/ml) for 16 h. Fig. 2A (left panel) shows the results of a representative experiment in which 30 min of metabolic labeling with [35S]methionine was followed by APP immunoprecipitation and gel electrophoresis (n 5 3). In the labeling shown, a 4-fold increase in the synthesis of intracellular APP was observed in response to a 16-h stimulus with IL-1a and a 3-fold increase in response to a 16-h stimulus with IL-1b (maximal 5.9-fold induction of APP synthesis by IL-1a for primary astrocytes (Table I)). The C-8 antibody immunoprecipitated two proteins, of 110 and 130 kDa, corresponding to the mature and immature glycosylated APP holoprotein (43). We used the rate of synthesis of other proteins as positive and negative controls to confirm that IL-1a and IL-1b specifically increase APP mRNA translation. IL-1a and IL-1b induced a 4-fold increase of H-ferritin synthesis in primary astrocytes (Fig. 2A, right panel), whereas the rate of astrocytic L-ferritin synthesis was unchanged in response to both IL-1a and IL-1b (Fig. 2A, right panel). The level of apoE protein synthesis was also determined and found to be unchanged after IL-1a or IL-1b stimulus, thus serving as an additional internal loading control to the L-ferritin for measuring specific increases in the relative rate of APP synthesis in astrocytes (n 5 3) (Fig. 2B). IL-1a increased the rate of total protein synthesis by 60% (maximal increase) in primary astrocyte cultures, as measured by trichloroacetic acid precipitation of labeled proteins from triplicate lysates (Table I). The action of iron chelation with 100 mM desferrioxamine generated a similar reduction of synthesis of both L-ferritin and H-ferritin in addition to APP (Fig. 2A). Iron as ferrotransferrin had no effect on the rate of either APP

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FIG. 2. IL-1a and IL-1b increased APP and H-ferritin subunit protein synthesis and APP(s) secretion in human primary astrocyte cells, whereas protein synthesis for apoE, b-actin, and L-ferritin subunit remained unchanged. Panel A. Left group, cells were treated for 16 h, pulse labeled for 30 min, and derivative lysates were immunoprecipitated with an antibody specific to the COOH terminus of APP (C-8). From left, first lane, unstimulated cells; second lane, 0.5 ng/ml IL-1a; third lane, 0.5 ng/ml IL-1b; fourth lane, 10 mM Fe2Tf; fifth lane, 100 mM desferrioxamine (Df). Right group, these lysates were immunoprecipitated with a ferritin antibody. Panel B, the culture medium was treated and pulse labeled as in panel A and immunoprecipitated with apoE antibody. Panel C, cells and matched controls were treated with 0.5 ng/ml IL-1a for 2, 6, and 16 h, pulsed labeled for 30 min, and derivative lysates were immunoprecipitated with C-8 antibody and a b-actin antibody. Panel D, culture medium from cells were treated for 16 h, pulse labeled for 120 min, and immunoprecipitated with an NH2-terminal APP antibody (R1736). First lane, unstimulated; second lane, 0.5 ng/ml IL-1b; third lane, 0.5 ng/ml IL-1a.

or ferritin protein synthesis in primary astrocyte cultures. The apparent coordinate regulation of the APP and ferritin genes is discussed below in terms of the presence of homologous translational regulatory sequences in their 59-UTRs (see Fig. 6). Further immunoprecipitations from two additional time course experiments (2, 6, and 16 h) showed that APP synthesis increased most sharply after 6 h of stimulus of primary astrocytes with the IL-1a (Fig. 2C). Our data showed that a 6-h stimulation with IL-1a induced a maximal 5.9-fold increase in APP synthesis (Table I). A 3.8-fold increase of APP synthesis was observed after a 16-h IL-1a stimulation. b-Actin protein synthesis was increased by 70% (maximum) relative to unstimulated astrocytes after IL-1a stimulation (Fig. 2C). 2 h of stimulation with IL-1a increased APP synthesis by only 40% relative to untreated cells, whereas b-actin synthesis changed

by 0.65-fold under the same conditions. We concluded that APP synthesis peaked at a time point between 6 and 16 h after the IL-1a stimulation of primary astrocytes. Total protein synthesis increased by 60% in response to IL-1a, as measured by trichloroacetic acid precipitation of labeled proteins from each lysate (Table I). Evidently, IL-1a increased APP synthesis by a margin 3.7-fold greater than the induction of total protein synthesis in primary astrocytes. IL-1 also induced a 1.7-fold increase in the secretion of APP (Protease-Nexin II or APP(s)) from primary human astrocytes as measured by direct scintillation counting of labeled immunoprecipitates (Fig. 2D). Densitometry of autoradiographs from an additional experiment revealed that IL-1b induced APP(s) synthesis by 2.6-fold. In these experiments the medium was collected after a 2-h pulse labeling with [35S]methionine and

Similarities in APP mRNA and Ferritin mRNA Translational Control immunoprecipitated using an NH2-terminal antibody (against amino acids 595– 611 of the APP). IL-1a also enhanced secretion of APP(s) into the medium, causing a smaller 25% accumulation of APP(s). Thus, the levels of both cell-associated and secreted APP (APP(s)) were increased by exposure of primary astrocytes to IL-1. Northern blot analysis was used to compare the possible action of IL-1a to increase the steady-state levels of APP mRNA (3 kb) in primary astrocytes over the same time course TABLE I Time course of IL-1a-dependent increase of APP synthesis for comparison with total protein synthesis in primary human astrocytes Shown are the fold inductions of protein synthesis, representative of two separate time courses. The data in Fig. 2C are included in Table I. Total protein synthesis was measured as trichloroacetic acid-precipitable counts (cpm 3 105 (n 5 6)). The relative increase in protein synthesis presented in Table I (third row) was calculated from the quantitation of trichloroacetic acid-insoluble counts as follows. First row, 1IL-1a (2 h) 5 113 cpm 6 21 cpm; 2IL-1a (2 h) 5 89 cpm 6 12.5 cpm. Second row, 1IL-1a (6 h) 5 272 cpm 6 17 cpm; 2IL-1a (6 h) 5 167 cpm 6 33 cpm. Third row, 1IL-1a (16 h) 5 179 cpm 6 62 cpm; 2IL-1a (16 h) 5 175 cpm 6 39 cpm. Hours of IL-1a stimulus

APP b-Actin Total protein

2

6

16

1.4 6 0.65 0.5 6 0.045 1.3 6 0.17

5.9 6 1.70 1.5 6 0.11 1.6 6 0.16

3.8 6 1.70 1.4 6 0.13 1.0 6 0.22

FIG. 3. Time course showing that APP mRNA expression was unchanged in response to 0.5 ng/ml IL-1a stimulation of human primary astrocytes. Sequential Northern blot hybridizations were performed with cRNA probes against ACT mRNA (upper), APP mRNA (middle), and GAPDH mRNA (lower). The molecular weights of each RNA are: APP mRNA, 3,500; ACT mRNA, 1,500; and GAPDH mRNA, 1,000.

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for IL-1a induction of APP synthesis (Fig. 3). We measured no increase in APP mRNA levels in primary astrocytes after 2, 6, or 16 h of IL-1a stimulation (n 5 4). As a positive control for effective IL-1a signal transduction, exposure of astrocytes to the cytokine caused a pronounced increase in the steady-state levels of ACT mRNA (1.5 kb of ACT mRNA), as has been demonstrated previously (.10-fold; n 5 4) (17). As Northern blot loading controls, steady-state levels of astrocytic GAPDH mRNA and 28 S rRNA were unchanged after IL-1a stimulation (Fig. 3). Our data also demonstrated that not only IL-1a stimulation, but also IL-1b stimulation leaves APP mRNA levels unchanged in primary astrocytes (n 5 4) (data not shown). Therefore, astrocytic APP gene expression by IL-1 is mediated by translational mechanisms. IL-1 Enhances APP mRNA Translation in Astrocytoma Cells—The effect of IL-1a and IL-1b stimulation on APP and ferritin protein synthesis was also measured in human astrocytoma (U373MG) cells. Fig. 4A shows a representative experiment in which APP synthesis was, respectively, increased 3and 5-fold after 16 h of IL-1a or IL-1b stimulation. As for primary astrocytes, protein synthesis of L-ferritin subunit was unchanged, thereby serving as an internal control for the specificity of induced APP synthesis (Fig. 4B). Both IL-1a and IL-1b increased the rate of H-ferritin synthesis by 4 –5-fold in U373MG astrocytoma cells (Fig. 4B). At the same time, IL-1a and IL-1b stimulation for 16 h caused a marked increase in the protein synthesis of mature and immature (glycosylated form) of ACT, as measured by a 30-min pulse labeling and immunoprecipitation (Fig. 4C). Northern blot experiments (Fig. 4D) showed consistently that IL-1a and IL-1b stimulation did not increase the steady-state levels of APP mRNA when standardized for RNA loading by GAPDH mRNA expression (n 5 4). As in primary astrocytes, IL-1 induction of APP synthesis in U373MG cells did not reflect the steady-state levels of APP mRNA, clearly indicating that IL-1 regulates APP gene expression at the translational level in astrocytoma cells. To confirm that IL-1a and IL-1b signaling invokes a well characterized transcriptional activation in astrocytoma cells (33), we demonstrated that both cytokines specifically increased the steadystate levels of ACT mRNA (Fig. 4D). Therefore, IL-1 signal transduction pathways in astrocytoma cells operate to increase ACT gene expression (ACT protein synthesis) at the level of ACT gene transcription but enhance APP gene expression at a level of enhanced APP mRNA translation. Maximal IL-1 induction of APP synthesis in U373MG cells was observed in dose-response experiments using concentrations of IL-1a and IL-1b varying by 2 orders of magnitude (0.05, 0.5, and 5 ng/ml IL-1a and IL-1b). For example, a 15-fold increase in APP synthesis was quantitated from immunopre-

FIG. 4. IL-1a and IL-1b increase APP synthesis but have no effect on APP mRNA levels in U373MG astrocytoma cells. Panel A, cells were treated for 16 h, pulse labeled for 30 min, and derivative lysates were immunoprecipitated with COOH-terminal directed APP antibody (C-8). From left, first lane, unstimulated cells; second lane, 0.5 ng/ml IL-1a; third lane, 0.5 ng/ml IL-1b. Panel B, the same lysates immunoprecipitated with a ferritin antibody. Panel C, ACT protein synthesis after 16 h of IL-1 stimulation of astrocytoma cells. First lane, unstimulated cells; second lane, 0.5 ng/ml IL-1a; third lane, 0.5 ng/ml IL-1b. Panel D, Northern blot hybridizations with cRNA probes complementary to APP mRNA, GAPDH mRNA, and ACT mRNA sequences. First lane, unstimulated cells; second lane, 0.5 ng/ml IL-1a; third lane, unstimulated cells; fourth lane, 0.5 ng/ml IL-1b. U373MG cells were stimulated with IL-1 for 16 h.

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Similarities in APP mRNA and Ferritin mRNA Translational Control

FIG. 5. APP synthesis in U373MG cells is induced in a doseand time-dependent fashion by IL-1a and IL-1b stimulation in the absence of change in the steady-state levels of APP mRNA. Panel A, dose-response experiment measuring APP synthesis. Cells were treated and lysates harvested and immunoprecipitated with COOH-terminal directed APP antibody (C-8) as described in Fig. 4. Left group: from left, first lane, unstimulated cells; second lane, 0.05 ng/ml IL-1a; third lane, 0.5 ng/ml IL-1a; fourth lane, 5 ng/ml IL-1a. Right group: from left, first lane, unstimulated cells; second lane, 0.05 ng/ml IL-1b; third lane, 0.5 ng/ml IL-1b; fourth lane, 5 ng/ml IL-1b. Panel B, dose-response experiment measuring the steady-state levels of APP mRNA. Northern blot hybridization was performed with cDNA probe complementary to APP mRNA sequences. Lane 1, unstimulated cells; lane 2, 0.05 ng/ml IL-1a; lane 3, 5 ng/ml IL-1a; lane 4, 0.05 ng/ml IL-1b; lane 5, 5 ng/ml IL-1b. Panel C, left group, time course experiment measuring APP synthesis. Cells were treated, and lysates were harvested and immunoprecipitated with COOH-terminal directed APP antibody (C-8) as described in Fig. 4A. From left, first lane, unstimulated cells; second lane, 0.5 ng/ml IL-1b stimulation for 2 h; third lane, 0.5 ng/ml IL-1b stimulation for 6 h. Right group, time course experiment measuring the steady-state levels of APP mRNA. Northern blot hybridization was performed using a labeled cDNA probe against APP mRNA. First lane, in vitro translated APP mRNA marker (3.5 kb); second lane, unstimulated cells; third and fourth lanes, 0.5 ng/ml IL-1b, 2 and 6 h, respectively.

cipitations of astrocytoma cells stimulated with 5 ng/ml IL-1a for 16 h (Fig. 5A). Two dose-response experiments showed that all three concentrations of IL-1a and IL-1b generated an average 12-fold increase of APP synthesis in U373MG cells (Table II, n 5 2). In the same experiments, IL-1 elevated total protein synthesis by only 2–3-fold in U373MG cells, as measured by [35S]methionine incorporation into trichloroacetic acid-insoluble counts (Table II). To confirm translational regulation, IL-1 stimulation did not increase APP mRNA levels over the same 0.05–5 ng/ml concentration range of IL-1a and IL-1b used to generate a 12-fold increase APP synthesis (Fig. 5B). Additional time course experiments (2 h and 16 h) with astrocytoma cells reflected closely the pattern of translational

regulation of APP gene expression by IL-1 observed in primary astrocytes (as shown in Fig. 2C). In U373MG cells, IL-1b (0.5 ng/ml) increased APP synthesis starting 6 h after cytokine stimulation (Fig. 5C, left panel). APP levels were unchanged after 2 h stimulation. By contrast, the steady-state levels of APP mRNA were unchanged at all time points after IL-1b stimulation (Fig. 5C, right panel). Multiple immunoprecipitation experiments demonstrated that IL-1a and IL-1b each generated an overall average 5-fold and 9-fold induction of APP synthesis in astrocytoma cells (n 5 7). These data confirm that 1) IL-1 regulates APP synthesis at the level of message translation in astrocytoma cells; that 2) IL-1-dependent translational regulation of astrocytoma APP mRNA begins after 6 h of cytokine stimulation, reflecting the induction profile of APP mRNA translation in primary astrocytes; and that 3) IL-1 induction of APP synthesis is 2–5-fold greater than the induction of total protein synthesis in astrocytoma cells. An IL-1-dependent Translational Enhancer in the APP mRNA 59-UTR—A consistent feature of our cell culture labeling experiments was that IL-1 and iron chelation with desferrioxamine generated a similar profile for APP and ferritin synthesis (Fig. 2). Previously, the 59-UTRs of the L-ferritin and H-ferritin genes (174 bp to 1142 bp from the L-ferritin gene cap site and 1139 bp to 1199 bp from the H-ferritin gene cap site) have been shown to confer both base-line and IL-1b-dependent translation to a CAT reporter gene transfected in human hepatoma cells (37). Therefore we aligned L- and Hferritin gene 59-UTR sequences with the APP gene. Fig. 6 shows the presence of an unexpectedly high 51% sequence homology sequence alignment between the L-ferritin and APP mRNA 59-UTRs (Gap program, Genetics Software from University of Wisconsin, Madison). Because the APP mRNA 59UTR contained a significant sequence homology to IL-1-responsive 59-UTR translational regulatory sequences in both L- and H-ferritin mRNAs (185 bp to 1 146 bp from the 59-cap site of the APP gene; Fig. 6), we tested whether these APP mRNA 59-UTR sequences could confer IL-1-dependent translation enhancement. A 90-nucleotide DNA fragment, encoding sequences from positions 1 55 to 1144 between the SmaI to NruI sites of the APP gene 59-UTR, was inserted immediately upstream of a hybrid CAT reporter gene. The resulting reporter construct was designated as pSV2(APP)CAT because it was a derivative of the pSV2CAT expression vector. Multiple transfection experiments with the pSV2(APP)CAT construct showed that both IL-1a and IL-1b, respectively, conferred an average 3-fold and 4-fold translational enhancement to CAT reporter mRNAs in U373MG astrocytoma cells (n 5 6) (Fig. 7B). Panel A shows a duplicate experiment where IL-1a increased CAT gene expression by 6-fold, and IL-1b increased CAT gene expression by 9-fold in pSV2(APP)CAT-transfected astrocytoma cells. This induction was sufficient to account for a significant proportion of the IL-1-enhanced APP synthesis in astrocytoma cells. As a negative control, IL-1b stimulation of pSV2CAT-transfected astrocytoma cells did not increase CAT activity, confirming that the APP mRNA 59-UTR is a translational regulatory element (36). In the representative experiment shown in Fig. 7C no sequences in the parental vector pSV2CAT conferred IL-1-dependent translational regulation. At the same time CAT activity was enhanced 3-fold in pSV2(APP)CAT transfectants. Northern blot analysis confirmed that a 16-h exposure to both IL-1a and IL-1b (0.5 ng/ml) did not significantly change the steady-state levels of the transfected APP/CAT hybrid mRNA in pSV2(APP)CAT-transfected astrocytoma cells (Fig. 7D). Purified RNA from either pSV2(APP)CAT or pSV2CAT (negative control) transfectants was hybridized to labeled antisense RNA sequences homolo-

Similarities in APP mRNA and Ferritin mRNA Translational Control

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TABLE II Dose-responsive increase of APP synthesis for comparison with total protein synthesis in U373MG cells (16-h stimulation with IL-1a and IL-1b) Shown is the average fold induction of APP synthesis as quantitated from densitometry from two separate dose-response inductions. The data shown in Fig. 2C are included in Table II. A maximum 15-fold induction of APP synthesis was observed (5 ng/ml IL-1a for 16 h). Total protein synthesis was measured as trichloroacetic acid-precipitable counts (cpm 3 105 (n 5 6)). The relative increase in protein synthesis was calculated after quantitation of trichloroacetic acid-insoluble counts as follows: 1, no stimulation 5 247 cpm 6 29 cpm; 2, IL-1a (0.05 ng/ml) 5 524 cpm 6 79 cpm; 3, IL-1a (0.5 ng/ml) 5 597 cpm 6 76 cpm; 4, IL-1a (5 ng/ml) 5 519 cpm 6 75 cpm; 5, no stimulus 5 190 cpm 6 75 cpm; 6, IL-1b (0.05 ng/ml) 5 560 cpm 6 18 cpm; 7, IL-1b (0.5 ng/ml) 5 602 cpm 6 18 cpm; 8, IL-1b (5 ng/ml) 5 412 cpm 6 29 cpm. IL-1a concentration (ng/ml)

Total protein (n 5 3) APP (n 5 2)

IL-1b concentration (ng/ml)

0.05

0.5

5.0

0.05

0.5

5.0

2.1 6 0.24 7.15

2.4 6 0.30 8.7

2.0 6 0.30 12

2.9 6 0.03 7.8

3.1 6 0.22 11

2.2 6 0.15 12.3

FIG. 6. Hybrid CAT constructs expressing the APP mRNA 5*-UTR. Upper panel, the pSV2(APP)CAT construct was prepared by inserting a 90-bp SmaINruI fragment of the APP gene 59-UTR immediately upstream of the CAT mRNA start codon. Computer alignment between the 59-UTR of the APP gene and the IL-1-responsive 59-UTR translational enhancer in L-ferritin mRNA revealed 51% sequence homology (bold lettering). The acute box homology motif is underlined (36). Lower panel, comparison of the predicted folding of RNA by computer analysis of the APP mRNA 59-UTR and the IL-1-responsive L-ferritin mRNA 59UTR translational enhancer (37). The APP mRNA acute box sequence is predicted to fold into a stable RNA stem-loop structure (47). This RNA stem-loop is identical to a larger stem-loop folded from the complete APP mRNA 59-UTR (DG 5 254 kCal/mol). The corresponding L-ferritin mRNA 59-UTR stem-loop, specific to the acute box consensus, folds into a less stable RNA structure (DG 5 216 kCal/mol).

gous to the 59-end of the CAT gene. The pSV2CAT transfectants expressed a 1,527-nucleotide CAT mRNA as expected (Fig. 7D, lanes 1 and 2, shows two separate loadings, 10 and 2 mg, respectively). The pSV2(APP)CAT-transfected cells expressed a closely migrating APP/CAT mRNA (1,617 nucleotides), larger by the presence of the 90-nucleotide insert coding for the APP gene 59-UTR, but also transcribed another larger (1,640 nucleotides) APP/CAT transcript (Fig. 7D, lane 3). This APP/CAT mRNA was likely the result of using a second poly(A) addition site downstream from the CAT gene stop codon in pSV2CAT.

IL-1a and IL-1b stimulated the reappearance of only the single 1,617-nucleotide APP/CAT mRNA transcript using the upstream poly(A) addition site. Densitometric quantitation of autoradiographs showed that IL-1a or IL-1b only modestly (30%) increased the total quantity of APP/CAT mRNA transcribed in pSV2(APP)CAT transfectants relative to standardizing GAPDH mRNA levels (Fig. 7D, lanes 3–5). Slot-blot analysis has convincingly confirmed that neither IL-1a nor IL-1b altered the steady-state levels of APP/CAT mRNA in pSV2(APP)CAT-transfected astrocytoma (U373MG) cells (data

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Similarities in APP mRNA and Ferritin mRNA Translational Control not shown). These measurements confirm previous reports characterizing IL-1-dependent translational regulation mediated by equivalent L- and H-ferritin 59-UTR acute box sequences (36, 37). Enhanced Base-line Translation by the APP Gene 59-UTR mRNA—Standardized transfections also revealed that baseline CAT gene expression was increased when APP gene 59UTR sequences were inserted between the unique HindIII and StuI sites in the 59-UTR of the CAT gene in pSV2CAT (Fig. 8). This effect accounts for the consistent difference in the baseline CAT gene expression derived from pSV2(APP)CAT compared with pSV2CAT in astrocytoma cells, as observed in Fig. 7C. In addition to mediating an IL-1-dependent increase in translation, the APP 59-UTR conferred a consistent 3– 4-fold increase in basal CAT activity in pSV2(APP)CAT-transfected U373MG cells compared with the parental pSV2CAT vector transfectants (Fig. 8, upper panel). The amount of CAT gene expression was standardized with the RSV2GAL plasmid (5 mg/transfection) to account for differences in transfection efficiency. In a separate study we have been examining the translational regulatory action of the APP gene 59-UTR in human neuroblastoma cells (SKN SH neuroblastoma cells). In addition to basal translational regulation in astrocyte-derived cells, multiple transfections of the pSV2(APP)CAT construct confirmed that the APP mRNA 59-UTR is also a basal translational regulatory element in neuroblastoma cells (n 5 6) (Fig. 8, lower panel). Increased base-line translational efficiency conferred by the APP mRNA 59-UTR was measured at 3.6-fold in astrocytoma cells (4.2 6 0.06-fold in neuroblastoma cells, n 5 6). DISCUSSION

FIG. 7. A 90-nucleotide SmaI to NruI sequence element from the 5*-UTR of APP mRNA confers IL-1-dependent CAT gene expression in U373MG cells. IL-1 did not change the steady-state levels of APP/CAT mRNA, and IL-1 did not change CAT gene expression in pSV2CAT transfected astrocytoma cells. Panel A, representative duplicate assay for CAT activity in lysates of pSV2(APP)CAT-transfected U373MG astrocytoma cells. From left, first and second lanes, unstimulated; third and fourth lanes, IL-1a-stimulated; fifth and sixth lanes, IL-1b-stimulated for 16 h. Panel B, average fold CAT activity conferred by the APP mRNA 59-UTR in response to IL-1a and IL-1b stimulation of astrocytoma cells from multiple separate transfections (mean 6 S.E.; n 5 6). Panel C, left group, representative experiment showing CAT activity in lysates of U373MG astrocytoma cells transfected with the pSV2CAT parental vector and treated for 16 h (n 5 6). First lane, unstimulated cells; second lane, 0.5 ng/ml IL-1b. Right group, astrocytoma cells transfected with the pSV2(APP)CAT vector and treated for 16 h. First lane, unstimulated cells; second lane, 0.5 ng/ml IL-1b. The lysates were normalized for transfection efficiency using 5 mg of a reference RSV2GAL plasmid. Panel D, Northern blot hybridizations of RNA purified from pSV2CAT and pSV2(APP)CAT-

This report provides the first evidence that IL-1 substantially induces APP synthesis in primary human astrocytes and astrocytoma cells by a mechanism of enhanced message translation. The translational efficiency of astrocytic APP mRNA was specifically and selectively enhanced by IL-1, while the translational efficiencies of the astrocytic mRNAs for b-actin, L-ferritin, and apoE were unaffected (H-ferritin synthesis is increased in astrocytes (Figs. 2A and 4B)). Induced APP synthesis was not observable after 2 h, but required 6 h of IL-1b stimulation in both primary astrocytes and in U373MG astrocytoma cells. IL-1 was shown to increase total astrocytoma protein synthesis by 2–3-fold, similar to insulin signaling of protein synthesis in HEK293 cells (44). The cytokine specifically induced a more substantial level of APP synthesis (Tables I and II). A similar time course of increased L- and H-ferritin mRNA translation during inflammation has been demonstrated in rat liver cells (45). The expression ratio of APP isoforms (APP-695:APP-751:APP-770) in astrocytes is 1:4:2 (i.e. the Kunitz-containing APP isoforms are the major APP isoforms in this cell type as reported previously), whereas APP695 predominates in neuronal cells (2, 42, 43). Both cytokine isoforms, IL-1a and IL-1b, increased APP synthesis, although IL-1b enhanced the secretion of APP(s) to a greater extent than IL-1a. Differences in the magnitude of

transfected astrocytoma cells (control and IL-1-stimulated) with a labeled cRNA probe complementary to the 59-end coding sequences and 59-UTR of the CAT gene from the pSBCAT subclone (36). Lanes 1 and 2, 10 and 2 mg of RNA from astrocytoma cells transfected with the parental vector (pSV2CAT); lanes 3–5, 10 mg of RNA from astrocytoma cells transfected with the pSV2(APP)CAT vector and treated for 16 h as follows: lane 3, unstimulated cells; lane 4, 0.5 ng/ml IL-1a; lane 5, 0.5 ng/ml IL-1b. The Northern blot shown was standardized for loading by use of a GAPDH gene probe. The ratio of IL-1 induction of APP/CAT to GAPDH mRNA established that IL-1 only increased the overall expression of transfected APP/CAT mRNA by a 30% margin.

Similarities in APP mRNA and Ferritin mRNA Translational Control

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FIG. 8. Top histogram, representative transfection experiment showing that the APP gene 59-UTR sequences conferred a 3.8-fold increased basal CAT gene expression in U373MG cells transfected with pSV2(APP)CAT compared with the parental pSV2CAT vector (ratio of 1.7 to 6.4% acetylation (n 5 2). Lower histogram, quantitation of multiple transfection experiments showing that the APP gene 59UTR sequences conferred 4.3-fold increased basal CAT gene expression in SKN-SH neuroblastoma cells transfected with pSV2(APP)CAT compared with the parental pSV2CAT vector (n 5 6). Differences in transfection efficiency were normalized using a reference RSV2GAL plasmid (mean 6 S.E. n 5 6).

cytokine-stimulated secretion of [35S]methionine-labeled APP(s) and nascent APP are likely the result of additional actions by IL-1 to alter the APP processing (1, 46). It has been shown previously that the effect of IL-1b on APP processing in human umbilical vein endothelial cells is mediated by the IL-1 receptor (46). The two cytokines are known to differ in a number of biological responses that they illicit (28). IL-1a is a cell-associated cytokine expressed as a fully active 31-kDa precursor protein (pro-L-1a) that is cleaved into a mature 17-Da IL-1a product. In contrast, IL-1b operates at the systemic level, where only the cleaved 17-Da cytokine is active. Additionally, only IL-1b preferentially binds to the IL-1 receptor II, perhaps also modifying signal transduction though the IL-1 receptor I (28). IL-1a does not bind at a high affinity to IL-1 receptor II. We identified a novel IL-1-responsive and basal translational enhancer in the 59-UTR of the APP gene, consistent with computer alignment with similar 59-UTR sequences in the ferritin genes. Ferritin gene expression has been well characterized and is known to be regulated at the level of message translation in hepatoma cells (37). Transfection studies with a hybrid

APP/CAT mRNA construct confirmed that the action of this sequence, mapping from 1 55 to 1 144 nucleotides from the APP mRNA 59-cap site, was sufficient to mediate the translational regulation of APP mRNA by IL-1 in U373MG cells, as measured by CAT reporter activity. In contrast, the steadystate levels of transfected hybrid APP/CAT mRNA was unchanged, similar to findings from parallel CAT reporter studies with the ferritin mRNA acute box elements (36). The most straightforward interpretation of our results is that IL-1 elevates APP mRNA translation through the action of an IL-1responsive stem-loop structure in APP mRNAs. Computer alignment showed that sequences in the 59-UTR of APP mRNAs are homologous, but not identical, to the acute box sequence of L-ferritin mRNA 59-UTR which confers IL-1-dependent translation specifically in hepatoma cells. The L-ferritin mRNA sequence differs from the APP mRNA sequence, likely explaining the lack of L-ferritin gene translational regulation by IL-1 in astrocytoma cells. The APP mRNA 59-UTR sequence is highly GC-rich (80%) and is predicted to fold into a single stable RNA stem-loop structure (DG 5 254 kCal/mol in

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Similarities in APP mRNA and Ferritin mRNA Translational Control

Fig. 6, lower) (47). There are striking overlaps in the regulation of the APP gene and the L- and H-ferritin genes, each of which encodes the subunits for the central iron storage protein shown in Fig. 2. First, ferritin is an acute phase reactant, and increased ferritin synthesis and concomitant iron sequestration are consistent with the anemia associated with the inflammation of chronic diseases (35, 45). Ferritin gene expression is regulated at the translational level in hepatoma cells (35). Here, the APP gene is also shown to be an acute phase reactant, regulated at the translational levels in astrocytes. Second, APP mRNA 59-UTR sequences confer significant IL-1-dependent and basal translational enhancement to activate CAT reporter gene expression in pSV2(APP)CAT- transfected astrocytoma cells. Similar hepatic translational regulation is conferred by the IL-1-responsive acute box RNA sequences in the L-and H-ferritin mRNA 59-UTRs (37). Like the ferritin genes, the APP gene 59-UTR maintains efficient translation of APP in both astrocyte-derived and neuroblastoma cells. The L- and H-ferritin gene 59-UTRs are organized into two regulatory sequences: an ironresponsive element at the 59-cap site, which is responsive to iron (48), oxidative stress (49), phorbol esters (50) and thyroid hormone receptor (51); and a downstream acute box sequence that is both a base-line and an IL-1-dependent translational regulatory element that works in an iron-dependent fashion (Fig. 4B) (35). The presence of similar translational regulatory sequences in the 59-UTRs of both APP mRNA and ferritin mRNA is consistent with the known role for metal binding, including copper and likely iron, as a part of the normal function of APP in cells (52). APP mRNA 39-UTR sequences regulate APP gene expression by modulating message stability in human peripheral blood mononuclear cells (53) and regulating message translation in Chinese hamster ovary cells (54). In addition, other studies have indirectly shown translational regulation of APP gene expression. The steady-state levels of APP protein in the rat cerebral cortex, meninges, and in primary astroglial, microglial, and neuronal cultures have been reported not to reflect APP mRNA levels (55). Furthermore, the relative levels of APP-695 (KPI2) and APP-751 (KPI1) mRNA and their proteins have been found to be discordant in human brain. Each transcript was approximately equally abundant, whereas KPI1 proteins predominated (.82%) and at elevated levels in the Alzheimer’s brain (56, 57). Several reports suggest a direct connection between increased APP levels and the development of AD pathogenesis. This increase might be linked to inflammatory mechanisms. 1) Down’s syndrome brains and trisomy 16 mice show increased APP levels beyond the 0.5-fold increase that would be expected by gene dosage (12). 2) Overexpression of APP in transgenic mice is necessary, even in the presence of FAD mutations, for sufficient Ab peptide production to lead to development of amyloid filament deposits and an Alzheimer’s-like pathology (59, 60). Furthermore, APP synthesis correlates with Ab peptide production in vivo (61). 3) Traumatic brain injury, a known risk factor for AD, increases IL-1 as well as APP immunoreactivity in rat brain (20, 32). 4) IL-1 injected into the rat cerebral cortex increases the steady-state levels of APP protein at the site of the lesion (62), and primary astrocytes have been shown to be a source of secreted Ab peptides (63). Overexpression of IL-1 by centrally located microglia has been shown to be associated even with early forms of amyloid plaques, the non-neuritic diffuse plaques, as well as being increased strikingly during plaque development (17, 18, 62). IL-1 has been suggested as a driving force for amyloid plaque maturation (62), perhaps mediated by signaling by the cytokine to astrocytes surrounding the plaque structures and subse-

quent induction of APP and ACT protein synthesis (64). However, published findings are not consistent about whether sporadic AD is associated with increased APP gene transcription, with reports of both increased (65) as well as decreased levels of APP mRNA (66). Our data provide substantial in vitro evidence for increased APP synthesis by enhanced message translation in response to IL-1 in astrocytes. IL-1 enhancement of APP synthesis in astrocytes suggests that the accumulation of Ab peptides into plaques during AD may be accelerated by a pattern of local protein synthesis in glial cells. This model of elevated local APP synthesis by a cytokine-mediated mechanism is consistent with increasing experimental epidemiological evidence linking the use of nonsteroidal anti-inflammatory drugs to the risk for AD pathology (58, 67). Acknowledgments—We thank Dr. D. Selkoe for helpful discussions and the use of the C-8 antiserum specific to the carboxyl-terminal of APP and the anti-APP(s) antibody (R1736). We also are grateful to Dr. K. Bridges, Dr. F. Bunn, and R. Handin of the Hematology Division, Department of Medicine, Brigham and Women’s Hospital for their support. J. T. R. also acknowledges Dr. John Growdon, Alzheimer’s Disease Research Center Massachusetts, and Dr. L. Thal of the Neurosciences and Education Research Foundation at the University of California, San Diego. REFERENCES 1. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Liebhaber, I., Koo, E. H., Schenk, D., Teplow, D. B., and Selkoe, D. J. (1992) Nature 359, 322–325 2. Gray, C. W., and Patel, A. (1993) Mol. Brain Res. 19, 251–256 3. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. 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